P820: POTENTIAL IMMUNOSUPPRESSIVE EFFECT OF EXTRACELLULAR VESICLES FROM PATIENTS WITH MULTIPLE MYELOMA
Authors: Shinya Hagiwara, Masaki Ri, Arisa Asano, Ayako Masaki, Yoshiaki Marumo, Kentaro Hirade, Takahiro Nakashima, Shiori Kinoshita, Tomotaka Suzuki, Tomoko Narita, Hirokazu Komatsu, Shinsuke Iida
Journal: HemaSphere (2023)

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Extracellular vesicles (EVs), mainly exosomes, play a role in cell-cell communication by carrying lipids, proteins, small RNAs and possibly DNA between cells. Recently, it has been reported that EVs are involved in MM-related processes including angiogenesis, osteolysis, immunosuppression and drug resistance. Moreover, there are several reports on the emerging role of EVs as new therapeutic agents for the treatment of MM, and their potential use as clinical biomarkers for early diagnosis and disease classification. However, the mechanistic role of EVs in MM is still largely unknown. Aims: In this study, we focused on immune-related effects of EVs in MM. In particular, we aimed to determine whether EVs have a suppressive effect on CD8+ T cells. Methods: First, the immunosuppressive effect of EVs derived from patients with MM was investigated. Blood plasma or serum samples were collected from patients newly diagnosed with MM (n=30) or from healthy donors (HD) (n=10). EVs were separated by the polymer precipitation method. Peripheral blood mononuclear cells (PBMCs) from HD were activated with anti-CD3 antibody (1 µg/mL) and interleukin-2 (IL-2) (100 U/mL) for 24 hours, and then co-cultured for a further 72 hours with EVs (100 µg/mL) separated from patients with MM or HD. We then monitored the altered expression of cell-surface immune checkpoint receptors (PD-1, TIGIT, LAG3) on CD8+ T cells by flow cytometry. Additionally, interferon γ (IFN-γ) was also evaluated using an intracellular cytokine staining technique. Next, the immunosuppressive effect of EVs from MM cell lines was investigated. 13 MM cell lines were cultured for 48 hours in RPMI-1640 supplemented with 10% exosome-depleted fetal bovine serum and 1% penicillin/streptomycin. EVs were separated from these supernatants by the polymer precipitation method. Using EV from each cell line, PBMCs were treated in the same way as above, and the level of expression of each immune checkpoint marker (PD-1, TIGIT, LAG3) was evaluated (3 samples for each EV). Student’s t-test and Dunnett’s test were used for statistical analysis, with p<0.05 considered statistically significant. Results: First, regarding effects of EVs from patient with MM, although the proportion of CD8+ cells expressing PD-1 was decreased by co-culture with HD-derived EVs, its expression on CD8+ cells was sustained by patient-derived EVs (28.2% vs 36.0%, p<0.01). In addition, a significant elevation of the number of cells expressing TIGIT and LAG-3 was observed when PBMCs were co-cultured with patient-derived vs HD-derived EVs (30.1% vs 27.7% for TIGIT and 9.3% vs 6.2% for LAG-3). Finally, patient-derived EVs suppressed the production of IFN-γ than HD-derived EVs (13.1% vs 15.2%, p<0.01). Next, the effects of EVs from 13 cell lines were tested. The proportion of PD-1+ cells in activated PBMCs was higher after co-culture with EVs derived from 6 of the 13 MM cell lines (AMO1, OPM2, XG7, SKMM1, NCI-H929, and NOP1, p<0.001 for each) than in cultures without EVs. A significant elevation of TIGIT+ and LAG3+ cells was also observed but only with EVs from 3 of the 13 MM cell lines (AMO1, OPM2, XG7, p<0.001 for each). Summary/Conclusion: EVs isolated from MM patients or cell lines induced a slight but significant increase in the proportion of CD8+ T cells expressing immune checkpoint markers in PBMCs from HD, suggesting a potential contribution to T-cell exhaustion. Further investigation of the mechanisms involved is needed. Keywords: CD8 T cells, Multiple myeloma